Several Citrus diseases have been shown to be caused by infection with pathogenic viruses (Derrick, K. S. and Timmer, L. W., Annu. Rev. Phytopathol., 38. 181-205 (2000)). One of the most important of these viruses is Citrus Tristeza Virus (CTV), a member of the Closterovirus group which induces serious disease syndromes in citrus. For example, CTV induces quick decline that causes the death of trees grafted on sour orange rootstock, and stem pitting of scion cultivars regardless of the rootstock used (Bar-Joseph et al., Annu. Rev. Phytopathol., 27. 291-316 (1989)). These diseases cause significant losses in the citrus industry worldwide.
In Brazil, citrus tristeza, first detected in 1937, destroyed millions of trees of sweet orange grafted on sour orange rootstocks. The problem was solved by exchanging sour orange rootstock with Rangpur lime rootstock. Today, more than 85% of the 200 million citrus trees in Brazil are grafted on Rangpur lime rootstock (Gimenes-Fernandes, N. and Bassanezi, R. B., Summa Phytopathologica., 27. 93 (2001)).
In 1999, a new citrus disease, named Citrus Sudden Death (CSD) was discovered in Brazil (Gimenes-Fernandes, N. and Bassanezi, R. B. Summa Phytopathologica., 27. 93 (2001)). This disease affects sweet orange (Citrus sinensis) grafted on Rangpur lime rootstock (Citrus limonia), and causes the death of trees within a few months after the symptoms manifest (Gimenes-Fernandes, N. and Bassanezi, R. B. Summa Phytopathologca., 27. 93 (2001)). Although the disease was first observed in the sweet orange/Rangpur lime scion/rootstock combination, it has also been observed in orange trees cvs. Hamlin, Natal, Valencia, Pera, and Rubi, all grafted onto Rangpur lime (Bassanezi et al., Phytopathol., 93. 4. 502-512 (2003)).
Plants with CSD symptoms present generalized leaf discoloration, partial defoliation, decreased number of young shoots and absence of internal shoots (Bassanezi et al., Phytopathol., 93. 4. 502-512 (2003)). As the symptoms become more pronounced, the disease progresses rapidly and leads ultimately, to the death of the plant. The physiological status of the plant is important for the disease progression, since the severity of the symptoms increase at high water demand (Gimenes-Fernandes, N. and Bassanezi, R. B. Summa Phytopathologica., 27. 93 (2001)). The root system of the symptomatic plants is severely damaged and dies quickly as the disease progresses. CSD is also characterized by the development of a strong yellow stain in the phloem of the Rangpur lime rootstock (Gimenes-Fernandes, N. and Bassanezi, R. B. Summa Phytopathologica., 27. 93 (2001)). The time between the appearance of the first visible symptoms in the canopy and the death of the plant ranges from 1 to more than 12 months depending on season and citrus variety (Bassanezi et al., Phytopathol., 93. 4. 502-512 (2003)).
The number of symptomatic trees in one affected area (north of São Paulo State and south of Triângulo Mineiro region, west of Minas Gerais State, Brazil), where the disease was originally found, increased from 500 in 1999 to more than 300,000 in February, 2002, and more than 1 million in June 2003 (Bassanezi, et al., Phytopathol., 93. 4. 502-512 (2003)); (Román et al., Plant Disease, 88. 5. 453-467 (2004)). The pattern of CSD dissemination is similar to that of quick-decline, a disease caused by certain CTV isolates that elicit a graft union incompatibility when infected sweet orange scions are grafted onto sour orange rootstocks (Bassanezi et al., supra) however, CSD affects several sweet oranges grafted on Rangpur lime, a rootstock/scion combination that is not affected by the CTV strains that causes quick-decline (Bassanezi et al., supra).
Based on the spatial and temporal patterns of CSD dissemination, it has been hypothesized that CSD may be caused by an insect-vectored pathogen, potentially a new, undescribed strain of CTV (Bassanezi et al., supra.) Alternatively, a new virus could be the causative agent of the CSD disease.
To test if CSD is caused by a variant strain of CTV or is caused by a new virus, a genomic approach using shotgun sequencing of genomic viral RNA that had been randomly reverse transcribed and cloned in a plasmid vector, was used to study the disease described herein. Bioinformatic tools were developed for the identification and assembly of viral sequences. Using this approach, it was possible to obtain a saturated database of viral sequences from individual trees.
Genomic viral RNA isolated from citrus trees symptomatic or asymptomatic for CSD were reverse transcribed and the first strand cDNA was used to construct random-primed cDNA libraries. Around 2,000 cDNA clones from each tree were sequenced and the sequences were analyzed using BLASTX, BLASTN, and TBLASTX searches against public databases. A viral genome assembled consensus sequence of 6820 nucleotides (SEQ ID NO: 1) encoding a viral polyprotein (SEQ ID NO: 2) sufficient to assemble a viral particle, was identified.
The 6820 nucleotides viral genome sequence, when translated in all possible frames, give rise to, at least, the following polypeptides: a Major Capsid Protein (Coat Protein 1) encoded by the Nucleotide Sequence Domain (NSD1) starting at nucleotide position 6028 and ending at nucleotide position 6675 of SEQ ID NO: 1, whose translation product give rise to the Amino Acid Sequence Domain (AASD) of the polypeptide of SEQ ID NO: 2, starting, at amino acid position 1974 and ending at amino acid position 2188; a Minor Capsid Protein (Coat Protein 2) encoded by NSD2 of SEQ ID NO: 1 starting at nucleotide position 6082 and ending at nucleotide position 6675, whose translation product give rise to the AASD of the polypeptide of SEQ ID NO: 2, starting at amino acid position 1992 and ending at amino acid position 2188; a Putative Movement Protein encoded by NSD3 of SEQ ID NO: 1 starting at nucleotide position 6260 and ending at nucleotide position 6724, whose translation product give rise to the AASD of the polypeptide of SEQ ID NO: 3, starting at amino acid position 1 and ending at amino acid position 154; a Methyltransferase Domain encoded by NSD4 of SEQ ID NO: 1 starting at nucleotide position 487 and ending at nucleotide position 1119, whose translation product give rise to the AASD of the polypeptide of SEQ ID NO: 2, starting at amino acid position 127 and ending at amino acid position 337; a Protease Domain encoded by NSD5 of SEQ ID NO: 1 starting at nucleotide position 2797 and ending at nucleotide position 3114, whose translation product give rise to the AASD of the polypeptide of SEQ ID NO: 2, starting at amino acid position 897 and ending at amino acid position 1002; a Helicase Domain encoded by NSD6 of SEQ ID NO: 1 starting at nucleotide position 3358 and ending at nucleotide position 4053, whose translation product give rise to the AASD of the polypeptide of SEQ ID NO: 2, starting at amino acid position 1084 and ending at amino acid position 1315; a RNA-dependent RNA polymerase encoded by NSD7 of SEQ ID NO: 1 starting at nucleotide position 4528 and ending at nucleotide position 5778, whose translation product give rise to the AASD of the polypeptide of SEQ ID NO: 2, starting at amino acid position 1474 and ending at amino acid position 1890.
The viral genome sequence showed strong similarity to several viruses from the Tymoviridae family of plant viruses, especially the oat blue dwarf virus (FIGS. 1 and 2). Analysis of CSD-symptomatic or asymptomatic trees for the presence of these viral sequences revealed that only trees presenting the CSD symptoms contain the viral sequences. It was therefore assumed that these sequences belong to an undescribed virus of the Tymoviridae family which is the causative agent of the CSD disease.